Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

doi: 10.4049/jimmunol.2100402

Figure Lengend Snippet: FIGURE 2. I3P boosts COX2 expression. (A) BMDMs were treated with 1 mM I3P for 1 h prior to stimulation with LPS (100 ng/ml) for 8 h. The cells were lysed, mRNA was extracted, and the expression of Ptgs2 was quantified by qPCR (n 5 3 from three independent experiments). (B) BMDMs were treated with 1 mM I3P for 1 h prior to stimulation with LPS (100 ng/ml) for 6 and 24 h. (C) COX2 was measured by Western blotting (n 5 4 from three independent experiments), and quantification by densitometry is shown. (D) BMDMs were treated with various concentrations (31.25 mM to 1000 mM) of I3P or control media for 1 h prior to stimulation with LPS (100 ng/ml) for 24 h. (E) COX2 was measured by Western blotting (n 5 5 from three independent experiments), and quantification by densitometry is shown. (FH) BMDMs were treated with 1 mM I3P for 1 h prior to stimulation with LPS (100 ng/ml) for 4, 6, or 8 h. The expression of Ptges (F), Pgis (G), and Tbxas1 (H) was measured by qPCR (n 5 3 from three independent experiments). All genes were normalized to rps18. (I) BMDMs were treated with 1 mM I3P for 1 h prior to stimulation with LPS (100 ng/ml) for 24 h. The concentration of PGs in both the cell lysates and supernatants was quantified by ELISA (n 5 2 from two independent experiments). Densitometry analysis is presented as mean ± SD. All other data are mean ± SEM. A one-way ANOVA was performed for (A), (C), and (E). A two-way ANOVA was performed for (F) and (G). The data show the adjusted p value obtained from multiple comparisons, corrected for using the Tukey test for one-way ANOVA or the Sidak test for two-way ANOVA. **p < 0.005, ***p < 0.0005, ****p < 0.0001.

Article Snippet: A total of 1 mg recombinant human COX2 (R&D Systems) was used per well, and for this experiment, DMSO was used as the solvent for I3P rather than cell culture media.

Techniques: Expressing, Western Blot, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

doi: 10.4049/jimmunol.2100402

Figure Lengend Snippet: FIGURE 3. I3P inhibits COX2 activity. (A) Schematic showing the structures of indole-3-acetic acid, indomethacin, and I3P. (B) BMDMs were treated with indomethacin (50 or 100 mM), I3P (0.5 or 1 mM), or the relevant vehicle control (DMSO or media) for 1 h prior to stimulation with LPS (100 ng/ml) for 24 h, and COX2 levels were measured by Western blotting (n 5 3 from three independent experiments). (C) Quantification by densitometry is shown. (D) Schematic depicting hypothesis of I3P inhibition of COX2. (E) BMDMs were treated with 1 mM I3P for 45 min, followed by addition of 5 mM AA or vehicle (DMSO) for a further 15 min, prior to stimulation with LPS (100 ng/ml) for 24 h. The concentration of PGs in the supernatants was quantified by ELISA (E) (n 5 5 from three independent experiments). (F) COX2 activity was measured after incubation of 1 mg recombinant COX2 with various concen- trations (31.25 mM to 1000 mM) of I3P or vehicle control (DMSO) (n 5 3 from three independent experiments). Densitometry analysis is presented as mean ± SD. All other data are mean ± SEM. A one-way ANOVA was performed for (C). A two-way ANOVA was performed for (E). The data show the adjusted p value obtained from multiple comparisons, corrected for using the Tukey test for one-way ANOVA or the Sidak test for two-way ANOVA. *p < 0.05, ****p < 0.0001.

Article Snippet: A total of 1 mg recombinant human COX2 (R&D Systems) was used per well, and for this experiment, DMSO was used as the solvent for I3P rather than cell culture media.

Techniques: Activity Assay, Control, Western Blot, Inhibition, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

doi: 10.4049/jimmunol.2100402

Figure Lengend Snippet: FIGURE 4. The augmentation of COX2 expression by I3P is regulated by a PG feedback loop. (A) BMDMs were treated with 10 mM PGE2 for 1 h and subsequently with 1 mM I3P for 1 h further, prior to stimulation with LPS (100 ng/ml) for 24 h. (B) COX2 was measured by Western blotting (n 5 3 from three inde- pendent experiments), and quantification by densitometry is shown. (C) BMDMs were treated with 5 mM of the EP2 antago- nist AH6809 for 1 h and subsequently with 1 mM I3P for 1 h fur- ther, prior to stimulation with LPS (100 ng/ml) for 24 h. (D) COX2 was measured by Western blotting (n 5 4 from three inde- pendent experiments), and quantification by densitometry is shown. (E) BMDMs were treated with 10 mM of the EP4 antago- nist GW 627368X for 30 min and subsequently with 0.5 mM I3P for 1 h further, prior to stimulation with LPS (100 ng/ml) for 24 h. (F) COX2 was measured by Western blotting (n 5 4 from three independent experiments), and quantification by densitome- try is shown. (G) BMDMs were treated with 10 mM forskolin for 30 min and subsequently with 1 mM I3P for 1 h further, prior to stimulation with LPS (100 ng/ml) for 24 h. (H) COX2 was mea- sured by Western blotting (n 5 4 from three independent experi- ments), and quantification by densitometry is shown. Densitometry analysis is presented as mean ± SD. A one-way ANOVA was per- formed. The data show the adjusted p value obtained from multiple comparisons, corrected for using the Tukey test. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001.

Article Snippet: A total of 1 mg recombinant human COX2 (R&D Systems) was used per well, and for this experiment, DMSO was used as the solvent for I3P rather than cell culture media.

Techniques: Expressing, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

doi: 10.4049/jimmunol.2100402

Figure Lengend Snippet: FIGURE 5. The augmentation of COX2 expression by I3P is partially dependent on AhR activation. (A) BMDMs from AhR1/1 and AhR/ mice were pretreated with 10 mM 3MC for 30 min prior to stimulation with LPS (100 ng/ml) for 24 h. (B) COX2 levels were measured by Western blotting (n 5 3 from one independent experiment), and quantification by densitometry is shown. (C) BMDMs from AhR1/1 and AhR/

Article Snippet: A total of 1 mg recombinant human COX2 (R&D Systems) was used per well, and for this experiment, DMSO was used as the solvent for I3P rather than cell culture media.

Techniques: Expressing, Activation Assay, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

doi: 10.4049/jimmunol.2100402

Figure Lengend Snippet: FIGURE 6. I3P blocks trypanosome lysate-induced PG production and LPS-induced PG production in human macrophages. (AC) BMDMs were pre- treated with 1 mM I3P or control media for 30 min before they were treated with 100 ng/ml IFN-g and 25 or 100 mg/ml trypanosome lysate for 24 h. COX2 expression was measured by Western blotting (n 5 3 from three independent experiments) (A), and quantification by densitometry is shown (B). (C) PGs were measured by ELISA (n 5 3 from three independent experiments). (DF) Primary human macrophages were pretreated with I3P (1 mM, 0.5 mM, or 0.25 mM) or control media for 1 h before they were treated with LPS (100 ng/ml) for 24 h. COX2 expression was measured by Western blotting (n 5 4 from three independent experiments) (D), and quantification by densitometry is shown (E). (F) PGs were measured by ELISA (n 5 4 from three independent experiments). (G) Schematic representation of inhibition of COX2 by I3P. Densitometry analysis is presented as mean ± SD. All other data are mean ± SEM. A one-way ANOVA was performed. The data show the adjusted p value obtained from multiple comparisons, corrected for using the Tukey test. ***p < 0.0005, ****p < 0.0001.

Article Snippet: A total of 1 mg recombinant human COX2 (R&D Systems) was used per well, and for this experiment, DMSO was used as the solvent for I3P rather than cell culture media.

Techniques: Control, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Inhibition